2017-4

Gencer EB, et al: Phenotypical Analysis of Umbilical Cord Blood
Turk J Hematol 2017;34:314-320
CFU Assays
CFU assays were implemented according to the manufacturer’s
recommendations (StemCell Technologies CFU Manual,
MA28404) and modified from Lee et al. [19]. First, 100 µL
of CB sample was removed from all CB units, and after the
addition of 80 µL of HetaSep and 300 µL of Iscove’s modified
Dulbecco medium containing 2% fetal bovine serum (StemCell
Technologies), the mixture was incubated at 37 °C in 5% CO 2
for 20 min (Sanyo CO 2
Incubator). Cells (5x10 5 cells/mL) were
transferred to 3 mL of MethoCult Express medium. After
14 days, colonies were counted and different morphologies
as well as numbers of CFUs were recorded using an inverted
light microscope (Olympus/IX51). The number of colonies was
calculated as the mean value for two dishes.
Statistical Analysis
Pearson correlation coefficient tests (if data distribution was
normal) and Spearman rank correlation coefficient tests (if data
distribution was not in the normal range) were used to assess
the correlations. All statistical analyses were performed with
SPSS 15.0.
Results
In this study we aimed to compare three different methods
in terms of efficiency to assess different re-populating HSCs
from CB, both after processing and after thawing. We analyzed
different cellular fractions, namely TNCs; CD34 + , ALDH + , CD34 +
ALDH + , ALDH + CD34 + , and ALDH + CD34 + CD90 + CD38 - cells; and
colony-forming units of granulocytes/macrophages (CFU-GM),
CFU-erythroids/granulocytes-macrophages/megakaryocytic
cells (GEMM), and burst forming units of erythroids (BFU-E),
for both fresh and post-thawed units. Table 1 demonstrates
the mean, median, and minimum-maximum values of the
aforementioned parameters for fresh and post-thawed samples.
Table 2 provides the correlation values for ALDH positivity and
TNC, CD34 + , and CD34 + CD90 + CD38 - cell numbers as well as
CFU-GM, CFU-GEMM, and BFU-E colony counts among all CB
samples. When fresh samples were analyzed, ALDH activity
correlated well with all the cell populations investigated; TNC,
ALDH + , and CD34 + fractions were found to be highly correlated
both with CFU-GM, CFU-GEMM, and BFU-E and with each other.
The correlation coefficients remained significant for fresh and
post-thawed samples, and when post-thaw data were analyzed,
TNCs, CD34 + , and ALDH were also found to be statistically
correlated with CFU-GM, CFU-GEMM, and BFU-E (Table 2).
Among all parameters compared, the most striking correlation
was detected for CFU-GM numbers and ALDH positivity for
fresh CB units (r=0.629, p<0.001); post-thaw analyses also
revealed a correlation for CFU-GM and ALDH + cells when the
same parameters were investigated (r=0.608, p=0.006; Table 2).
When CFU-GM numbers were tested against all parameters for
Table 1. Characteristics of all fresh and post-thaw CB units tested.
Variables (fresh CB units, n=30; post-thaw CB units, n=19) Mean ± SD Median Minimum - maximum
TNC
(x10 7 /U)
CD34 +
(x10 6 /U)
ALDH +
(x10 6 /U)
ALDH + /CD34 +
(x10 6 /U)
CD34 + /ALDH +
(x10 6 /U)
ALDH + /CD90 + /CD34 + /CD38-
(x10 6 /U)
CFU-GM
(x10 5 /U)
CFU-GEMM
(x10 5 /U)
BFU-E
(x10 5 /U)
Fresh 93.8±30.1 85.5 46.8-168.8
Post-thaw 32.79±17.27 31.05 9.2-47.6
Fresh 3.85±2.55 3.6 0.94-11.64
Post-thaw 2.18±3.17 1.18 0.5-14.5
Fresh 3.14±2.55 2.6 0.12-8.48
Post-thaw 2.01±2.81 1.38 0.20-11.3
Fresh 2.97±2.02 2.4 0.11-8.12
Post-thaw 1.82±2.63 1.28 0.17-1.69
Fresh 3.72±2.28 2.9 0.34-8.94
Post-thaw 3.01±4.33 1.65 0.29-15.31
Fresh 0.19±0.19 0.1 0.005-0.795
Post-thaw 0.40±0.55 0.2 0.01-1.92
Fresh 2.64±1.96 2.14 0.25-7.67
Post-thaw 0.74±0.92 0.41 0.02-2.92
Fresh 3.86±2.73 3.6 0.66-11.49
Post-thaw 0.70±0.98 0.36 0-4.27
Fresh 5.30±3.53 5.6 0.12-16.22
Post-thaw 0.44±0.74 0.19 0-2.9
SD: Standard deviation, CB: cord blood, TNC: total number of nucleated cells, ALDH: aldehyde dehydrogenase, CFU-GEMM: colony-forming units - granulocytes-macrophages/
megakaryocytic cells, BFU-E: burst forming units of erythroids, GM: granulocytes/macrophages.
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